PROJECT SUMMARY Increased reactive oxygen species (ROS) have been considered to be the main pathogenic factors in the development and progression of vascular dysfunction in diabetes. However, the mechanisms of ROS-induced microvascular complications and the interplay of ROS with nitric oxide (NO) and reactive nitrogen species (RNS) under diabetic conditions remain poorly understood. Currently, ROS-induced endothelial NO synthase (eNOS) uncoupling and NO deficiency-mediated vascular dysfunction have been extensively studied in cultured endothelial cells and arterioles. Very little is known about the direct effect of ROS on eNOS activity and permeability in venules, a crucial site for solute and fluid exchange and a major site of inflammation. Our preliminary studies conducted in intact rat venules revealed the roles of H2O2 in eNOS activation, NO production, peroxynitrite formation, and cellular and molecular mechanisms of H2O2-mediated permeability increases. Our findings that diabetic rats have increased plasma H2O2 and decreased catalase activity suggest that the mechanisms of H2O2-mediated changes in microvascular permeability may resemble those involved in ROS-mediated microvessel complication in diabetes. We hypothesize that ROS do not reduce NO production, but rather cause excessive NO production and peroxynitrite formation in venules. The NO-derived peroxynitrite further activates eNOS, resulting in augmented peroxynitrite formation. This self-promoting mechanism is the key for H2O2-induced peroxynitrite-mediated cell injury, Ca2+ overload in endothelial cells, and microvascular barrier dysfunction. The hypothesis will be tested in three specific aims: 1) investigate the cellular mechanisms of H2O2-induced NO production and NO-mediated microvascular barrier dysfunction; 2) investigate the role of NO-derived peroxynitrite in H2O2-induced microvascular barrier dysfunction; and 3) investigate the cellular and molecular mechanisms of ROS-mediated microvascular dysfunction in diabetes. The designed experiments with combined quantitative measurements of microvessel permeability along with confocal and electron microscopic investigation in individually perfused microvessels enable ROS-mediated changes in signaling molecules, enzyme activities, and vascular structures to be directly linked with changes in vascular barrier function. The addition of newly developed Nrf2 knockout rats that genetically modify antioxidant defenses into the proposal will benefit the mechanistic investigations of ROS-mediated microvascular complications in diabetes. The results derived from this proposal will provide new information that bridges studies using whole animals, organs, or vascular beds with studies using cultured endothelial cells and provide a better understanding of the pathogenesis of diabetes-associated microvascular complication and benefit the development of targeted therapeutics.